The high importance of signaling via heterotrimeric G proteins in physiology and disease is reflected by the fact that G protein-coupled receptors (GPCRs), which activate G proteins, are the target for more that >25% of FDA-approved drugs. Interestingly, recent work by us and others has led to the identification a novel mechanism of trimeric G protein activation that is mediated by cytoplasmic, non-receptor proteins instead of membrane-bound GPCRs. Although this mechanism has important implications in human disease and targeting it is a novel opportunity to develop therapeutic approaches, it remains completely unexploited form a pharmacological standpoint. Our goal is to carry out proof-of- concept studies for early stage drug development of first-in-class small molecule inhibitors of a GPCR- independent mechanism of G protein activation that promotes cancer metastasis. More specifically, our efforts will be focused on developing and characterizing small molecules that disrupt the protein-protein interaction (PPI) formed between the G protein G?i and its non-receptor activator GIV (aka Girdin). Many independent studies have demonstrated that GIV is upregulated in metastatic carcinomas. Upon GIV overexpression, the GIV-G?i PPI enhances signaling responses that lead to increased tumor cell migration and invasion. Thus, inhibition of the GIV-G?i PPI is a vulnerability of metastatic tumor cells that might provide a therapeutic window to treat aggressive metastatic cancers. This is of paramount significance because metastasis is the cause of >90% of cancer related deaths and there are very limited therapeutic options for it. PRELIMINARY DATA AND EXPERIMENTAL PLAN: In recently published work, we have characterized the structure of the GIV-G?i interface and demonstrated that it can be disrupted by small molecules. We have subsequently performed a screen of 200,000 compounds. We confirmed hits identified in the primary screen with an orthogonal biochemical assay, filtered out compounds with chemical liabilities and validated them analytically after re-purchase/re-synthesis. After evaluation in biological assays, we have identified small molecules based on four unrelated, synthetically tractable scaffolds that disrupt the GIV-G?i PPI with IC50's in the ~0.5-30 M range and that display the expected biological activity of blocking tumor cell migration without undesired non-specific toxicity. We hypothesize that these compounds selectively disrupt the GIV-G?i PPI to block the signaling and cell behavioral processes by which this PPI drives tumor invasiveness, and that upon optimization, these compounds will have therapeutic effects in pre-clinical models of metastasis. In Aim 1 we will comprehensively characterize the mode of action of the newly identified inhibitors using biochemical, biophysical and cell-based approaches, while in Aim 2 we will optimize the most promising of our compounds to increase its potency and druglike properties to achieve therapeutic effects in mouse models of metastasis.